Molecular Conformation And Substrate Specificity Of Mutant Imide Hydrolases

Cyclic imide hydrolase (CIH, EC.3.5.2.16), widely distributed inbacteria, yeast and molds, is a member of the cyclic amidase family.Nowadays, there are three reports about CIH, from Blastobacter sp. A17p-4,which was first purified and characterized, from Alcaligenes eatrophus112R4 which was first isolated and expressed in E.coli and fromPseudomonas putdia YZ-26 which was firstly reported in our laboratoryrespectively. CIH is a homologous tetramer with the subunit molecular massof 35 kDa, and 293 residues. CIH293 exhibits the higher activity and affinitytoward the simple cyclic imide such as succinimide. In bacteria, the enzymeis involved in catalyzing the ring-opening hydrolysis of cyclic imide tohalf-amide acid and then cleaving the dicarboxylic acid by the half-imidase.Finally, the end product enters TCA cycle.To explore the effect of C-terminal region residues on the molecularconformation and enzymatic activity of CIH293, several mutants, truncatedor substituted at C-terminus, were designed on the basis of its gene sequence.DNA fragments of intact CIH293 and its mutants were amplified by PCRwith plasmid pE-cih₂₉₃ as the template in the presence of different primers,and in turn, the amplified genes were subcloned into vector pE32M to formcorresponding recombinant plasmids. Recombinant plasmids then wereintroduced into E.coli BL21 (DE3), after induced expression, the enzymaticactivity of engineered strains was determined toward hydantoin as a routinesubstrate. Compared with CIH293, the enzymatic activity of mutants wasobviously decreased. The activity recovery is 78% for CIH292, 75% forCIH291 and 15% for CIH290 as well as 80% for KK292, 293EE, and 7% forKK292,293LL respectively. Based on the data of enzyme activity, the intactCIH293 and its mutants (CIH291, CIH290, KK292,293EE, KK292,293LL)were selected and purified to homogeneity by Ni²⁺-NTA agarose affinitycolumn and Sephacryl S-200 size-exclusion chromatography. In the view of enzymatic activity, polymeric state, CD and fluorescencespectra, the CIH activity was lost more and more with decreasing theresidues at C-terminus, while the molecular form was maintained as thehomotetramer. In addition, when the last two LysLys at C-terminus of theenzyme were substituted by GluGlu, there was no any obvious change bothon the conformation and activity, while the last two residues were substitutedby LeuLeu, the enzymatic activity was almost lost and the enzyme wasdissociated to be monomer. It means from above data that the positive chargeat C-terminus plays an important role in maintaining the structure andfunction of the CIH.The substrate specificity and kinetic parameters of these mutants weredetermined by both the photometric assay and reverse-phasehigh-performance liquid chromatography (RP-HPLC). For HPLC, thesubstrates and the corresponding products were separated through apre-packed Symmetry^? C₁₈ column (4.6mm×150mm) and monitored byUV-Vis detector on a Waters 1525 HPLC system. The separation wasachieved with the mixture of water and methanol as the mobile phase at aflow-rate of 1 mL/min. It is shown from both the photometric assay andHPLC that the substrate specificity of mutants has no any obvious change,but the affinity between the enzyme and its substrates is slightly lower,compared with the intact enzyme, CIH293.